As trypanosomes progress from the mammalian host through the tsetse fly, they encounter a series of distinct microenvironments that require specific and dynamic changes in their transcriptomes. Within the fly, these parasites exist as asynchronous, heterogeneous populations. To capture the complexity of these developmental transitions—previously obscured by bulk transcriptomics—several recent studies have leveraged single-cell technologies. These efforts have revealed: (a) putative ‘early regulators’ that commit cells to differentiation (Briggs et al, 2023), (b) novel surface proteins expressed in mature metacyclic forms (Vigneron et, al 2020), (c) simultaneous expression of multiple VSGs in pre-metacyclics, suggesting a transcriptional race to initiate mVSG expression in the salivary glands (Hutchinson et al, 2021), and (d) the transcriptional profiles of meiotic intermediates and gametes (Howick et al, 2022).
Building on this, we are applying single-cell functional genomics to investigate three gene cohorts previously implicated in T. brucei differentiation in vitro. We have developed RNAi libraries targeting 397 genes, enabling us to identify those required for parasite development within the fly. Our goals are to understand: (i) the developmental progression of trypanosomes within the tsetse fly, (ii) how individual cells differentiate as they move between tissue types, and (iii) the gene regulatory networks that orchestrate these transitions.
